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The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not completely understood. SARS-CoV-2 infection frequently causes significant immune function consequences including reduced T cell numbers and enhanced T cell exhaustion that contribute to disease severity. The extent to which T cell effects are directly mediated through infection or indirectly result from infection of respiratory-associated cells is unclear. We show that primary human T cells express sufficient levels of angiotensin converting enzyme 2 (ACE-2), the SARS-CoV-2 receptor, to mediate viral binding and entry into T cells. We further show that T cells exposed to SARS-CoV-2 particles demonstrate reduced proliferation and apoptosis compared to uninfected controls, indicating that direct interaction of SARS-CoV-2 with T cells may alter T cell growth, activation, and survival. Regulation of T cell activation and/or turnover by SARS-CoV-2 may contribute to impaired T cell function observed in patients with severe disease.

The COVID-19 pandemic has caused a high demand for respiratory protection among health care workers in hospitals, especially surgical N95 filtering facepiece respirators (FFRs). To aid in alleviating that demand, a survey of commercially-available filter media was conducted to determine whether any could serve as a substitute for an N95 FFR while held in a 3D-printed mask (Stopgap Surgical Face Mask from the NIH 3D Print Exchange). Fourteen filter media types and eight combinations were evaluated for filtration efficiency, breathing resistance (pressure drop), and liquid penetration. Additional testing was conducted to evaluate two filter media disinfection methods in the event that the filters were reused in a hospital setting. Efficiency testing was conducted in accordance with the procedures established for approving an N95 FFR. One apparatus used a filter-holding device and another apparatus employed a manikin head to which the 3D-printed mask could be sealed. The filter media and combinations exhibited collection efficiencies varied between 3.9% to 98.8% when tested with a face velocity comparable to that of a standard N95 FFR at the 85 L min(−1) used in the approval procedure. Breathing resistance varied between 10.8 to >637 Pa (1.1 to > 65 mm H(2)O). When applied to the 3D-printed mask efficiency decreased by an average of 13% and breathing resistance increased 4-fold as a result of the smaller surface area of the filter media when held in that mask compared to that of an N95 FFR. Disinfection by dry heat, even after 25 cycles, did not significantly affect filter efficiency and reduced viral infectivity by > 99.9%. However, 10 cycles of 59% vaporized H(2)O(2) significantly (p < 0.001) reduced filter efficiency of the media tested. Several commercially-available filter media were found to be potential replacements for the media used to construct the typical cup-like N95 FFR. However, their use in the 3D-printed mask demonstrated reduced efficiency and increased breathing resistance at 85 L min(−1).

As SARS-CoV-2 variants emerge, there is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines. A reference reagent comprised of post-vaccination sera from recipients of different vaccines allows evaluation of in vitro variant neutralization, and provides a reference for comparing assay results across laboratories. We prepared and pooled >1 L serum from donors who received the SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), a replication-incompetent adenovirus type 26 vaccine (Ad26.COV2.S, Johnson and Johnson), or recombinant spike protein expressed by baculovirus incorporated into a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). Twice frozen sera were aliquoted and are available for distribution to the research community (BEI Resources). The calculated WHO titer of pooled sera to spike protein was 1,312, 1,447, 1,936, and 587 and the reciprocal RBD binding to ACE-2 IC90-titers were 60, 64, 118, and 46 for BNT162b2, mRNA1273, Ad26.CoV2373, and NVX-CoV2373 sera, respectively.

Air pollution particulate matter (PM) is associated with SARS-CoV-2 infection and severity, although mechanistic studies are lacking. We tested whether airway surface liquid (ASL) from primary human airway epithelial cells is antiviral against SARS-CoV-2 and human alphacoronavirus 229E (CoV-229E) (responsible for common colds), and whether PM (urban, indoor air pollution [IAP], volcanic ash) affected ASL antiviral activity. ASL inactivated SARS-CoV-2 and CoV-229E. Independently, urban PM also decreased SARS-CoV-2 and CoV-229E infection, and IAP PM decreased CoV-229E infection. However, in combination, urban PM impaired ASL’s antiviral activity against both viruses, and the same effect occurred for IAP PM and ash against SARS-CoV-2, suggesting that PM may enhance SARS-CoV-2 infection., Primary human airway surface liquid (ASL) inactivates severe acute respiratory syndrome coronavirus 2 and human alphacoronavirus coronavirus 229E in vitro. Air pollution particulate matter (PM) inhibits ASL viral inactivation, suggesting PM may enhance coronavirus infection by impairing airway innate immunity.

Background: A Src kinase-activating phosphatase (PTPRE) is targeted by a genome-derived yellow fever virus (YFV) short noncoding RNA (vsRNA) in vitro. The vsRNA reduces PTPRE translation, which leads to reduced TCR signaling. vsRNA point mutations restore PTPRE expression and T cell function. We examined TCR signaling and PTPRE levels in individuals before and after YFV vaccination (YFVax). Methods: Fourteen individuals receiving YFVax (104.7–5.6) IM for travel prophylaxis provided written informed consent for these studies. Blood was obtained once before vaccination and four times after vaccination (days 3 to 28). Serum and PBMCs were purified and YFV was quantified by RNA and infectivity. PBMCs were assessed for activation following anti-CD3 stimulation by measuring phospho-tyrosine-394-Lck and IL-2 release. PBMC PTPRE levels were determined by immunoblot analyses (normalized to actin). A YFV-neutralizing antibody was determined by PRNT. Results: YFVax was administered alone (six out of 14 subjects) or in combination with other vaccines (eight out of 14). All subjects demonstrated reduced resting PBMC PTPRE levels and post-TCR stimulation had reduced IL-2 release between days 4 and 21 compared to pre- and day 28 samples. Phospho-Lck was reduced in all but two subjects on the same days, and both of these subjects also received an influenza vaccine. Low-level viremia was detected in 10/14 subjects, with infectious titers of 100/mL. Viremia was not detected in four out of 14 subjects. All recipients developed neutralizing antibodies by day 21. Conclusion: YFV vaccination regulates PBMC PTPRE levels 4–21 days after infection, despite the low to absent infectious YFV detected in serum, suggesting that enough YFV vsRNA is produced and released from cells to have a functional (and measurable) effect on T cell function. Studies are underway to determine if this is mediated by exosomes or defective particles containing the vsRNA that targets PTPRE. Furthermore, the association between PTPRE and TCR signaling confirms a role for PTPRE in TCR function.

Background: Program death receptor 1 (PD-1) is a co-inhibitory receptor that is upregulated and contributes to T cell dysfunction (exhaustion) during chronic viral infections, including HIV and HCV. GB virus C (GBV-C) is a persistent human virus, and co-infection is associated with reduced immune activation and improved clinical outcomes in HIV- and Ebola-infected individuals. Methods: PD-1 levels were measured by flow cytometry on CD38+ T cells from 45 HIV-infected individuals, 20 of whom were co-infected with GBV-C. Jurkat cell lines that stably express GBV-C E2 protein and vector control were used to purify total cellular RNA before, and 24 h following, activation using anti-CD3/CD28 treatment. Gene expression was analyzed by RNA-seq and qRT-PCR. Results: HIV-infected individuals with GBV-C viremia had reduced PD-1 expression on activated CD4+ and CD8+ T cells compared to HIV-infected GBV-C negative individuals. GBV-C particles and GBV-C E2 protein each inhibited PD-1 expression on T cells in vitro. Consistent with this, GBV-C E2 reduced gene expression of PD-1, and its ligand PD-L1, in both resting and activated T cells. GBV-C E2 regulated transcription of the PD-1 signaling pathway and T cell activation associated genes, without downregulation of the interferon-stimulated and innate immunity-related genes needed to resolve viral infections. Conclusions: Our current understanding of chronic RNA virus infections is that upregulation of PD-1 with T cell exhaustion is critical for viral persistence. However, these data demonstrate that GBV-C infection reduced PD-1 expression on activated T cells during HIV infection, and that the GBV-C E2 protein inhibits PD-1 signaling in T cells. This may preserve T cell function and contribute to the lack of immune deficiency in people with chronic GBV-C infection. Understanding the mechanisms by which GBV-C E2 alters PD-1 signaling may aid in the development of novel immunomodulatory therapeutics to prevent T cell dysfunction (exhaustion) during chronic viral infections.

Background: We previously identified a yellow fever virus (YFV) genome-derived, short noncoding RNA (vsRNA) generated during infection that targets the 3’ untranslated region (UTR) of a Src-kinase regulatory phosphatase (PTPRE), reducing PTPRE translation. By contrast, a related flavivirus (Zika) did not regulate PTPRE. We assessed the role of PTPRE in YFV and Zika replication. Methods: Human PTPRE with a β -Globulin 3’UTR to remove the 3’UTR sequence targeted by the YFV vsRNA was stably expressed in Jurkat and Huh7D cells using a tetracycline-regulated promoter. YFV (17D strain) and Zika (PR strain) were used to infect the different cell lines, and viral replication was measured by viral RNA and infectivity. Attempts to knock out PTPRE by CRISPR were unsuccessful, suggesting that PTPRE may be required for cell survival. Results: The expression of PTPRE with nontargeted 3′UTR led to a 100-fold reduction in infectious virus released from Jurkat and Huh7D cells by infectivity and a 3–10-fold reduction in viral RNA levels. Infectivity was restored in cells grown in doxycycline. By contrast, nontargeted PTPRE expression had no effect on the release of infectious Zika virus. PTPRE expression did not alter YFV attachment or entry. Despite reduced infectivity of the supernatant virus produced by PTPRE-nontargeted cells, YFV envelope content in culture supernatants was similar to that released by cells not expressing nontargeted PTPRE. Electron microscopy demonstrated large, empty-appearing viral particles in YFV-infected cells expressing nontargeted PTPRE that were not present in cells not expressing nontargeted PTPRE. Conclusions: PTPRE is a novel YFV restriction factor that appears to increase the proportion of defective YFV particles released into cell culture media, potentially by preventing YFV RNA encapsidation. PTPRE’s effects on viral replication are not conserved among flaviviruses, as Zika replication was not altered by PTPRE overexpression. Understanding how PTPRE inhibits YFV may provide insight into YFV assembly and release and potentially help to identify novel drug targets.

To understand the thermal performance of bamboo scrimber at elevated temperatures, its specific heat, density ratio and conductivity were measured using TGA-DSC and Hot Disk. With temperature increasing from 20 °C to 100 °C, the specific heat and conductivity of bamboo increase gradually. The density ratio decreases to about 0.25 at 500 °C and stays constant until 900 °C. The conductivity of bamboo scrimber parallel to grain is 1.26–1.63 times the conductivity perpendicular to grain. Due to the high density and content of phenolic resin, the conductivity of bamboo scrimber is higher than those of glued laminated timber and timbers in Eurocode 5 and the literature. Charring models of bamboo scrimber parallel to grain and perpendicular to grain exposed to one-sided fire were simulated and compared with experimental results. The relative difference in charring depth is less than 10%. A 3D finite element model of bamboo scrimber beams exposed to four-sided fire was established based on the measured thermal parameters. The results indicate that the measured specific heat, density ratio and conductivity of bamboo scrimber have good accuracy and are suitable for numerical simulation. Therefore, the reliable thermal parameter data can be used in fire resistance analysis.

At present, the artificial fruit picking cost is high, the fruit damage rate is high, and the efficiency is low, so the greenhouse picking robot emerges as the times’ demand. Therefore, this paper designs a kind of a chassis system of greenhouse picking robots. This paper mainly designed and simulates the chassis mechanical system, electrical system, and basic motion control algorithm from three aspects. Finally, the prototype of the chassis system of the picking robot in the greenhouse is completed and the chassis system is debugged, which proves that it has the characteristics of adaptability to the greenhouse environment, strong universality, and strong expansibility.

To understand the stress-strain relationship and failure mechanism of bamboo scrimber at elevated temperatures, both parallel and perpendicular to grain properties in tension and compression at temperatures from 20 °C to 270 °C were experimentally studied. A total of 90 tensile coupon specimens and 60 compressive specimens were tested during heating at temperatures ranging from 20 °C to 270 °C. The experimental results indicated that tensile stress-strain relationships of a bamboo scrimber parallel to grain at elevated temperatures were linear from the beginning of loading to failure, and the tensile strength and modulus decreased with increasing the temperature from 20 °C to 270 °C, except at 200 °C. Perpendicular to grain direction, the tensile stress-strain relationships exhibited nonlinear behaviour, and the ultimate strengths were very small. Accordingly, it was observed that the modulus decreased to zero from 20 °C to 180 °C. The compressive stress-strain curves of bamboo scrimber exposed to elevated temperatures were divided into a linear branch and a nonlinear branch beyond proportional limit for both grain directions. The equations with reduction factors for strength and modulus of elasticity were established by regression analysis of the test data. Parallel to grain of a bamboo scrimber, the values from fitting formulas were mostly larger than those from Eurocode 5, and the maximum differences between reduction factors were 0.31, 0.04, 0.23, and 0.24. This study provides a valuable reference for potential application of bamboo scrimber in structural engineering.

Background Human pegivirus (HPgV) is a single-strand RNA virus belonging to the Flaviviridae. Although no definitive association between HPgV infection and disease has been identified, previous studies have suggested an association of HPgV viremia with risk of lymphomas. Methods We conducted a systematic review and meta-analysis, including 1 cohort study and 14 case-control studies, assessing the association of HPgV viremia with adult lymphomas. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a random-effects model, overall and by geographic region and lymphoma subtype. Results The overall OR for lymphoma was 2.85 (95% CI, 1.98–4.11), with statistically significantly elevated ORs observed in 8 of 15 studies. There was a small amount of heterogeneity among studies (I2 = 28.9%; Q = 18.27, P = .16), and the funnel plot provided no evidence for publication bias. The strongest association with lymphoma risk was observed for studies from Southern Europe (OR, 5.68 [95% CI, 1.98–16.3]), whereas weaker ORs (with 95% CIs) were observed for studies from North America (2.24 [1.76–2.85]), Northern Europe (2.90 [.45–18.7), and the Middle East (2.51 [.87–7.27]), but all of similar magnitude. Participants with HPgV viremia had statistically significantly increased risks (OR [95% CI]) for developing diffuse large B-cell (3.29 [1.63–6.62]), follicular (3.01 [1.95–4.63]), marginal zone (1.90 [1.13–3.18]), and T-cell (2.11 [1.17–3.89]) lymphomas, while the risk for Hodgkin lymphoma (3.53 [.48–25.9]) and chronic lymphocytic leukemia (1.45 [.45–4.66]) were increased but did not achieve statistical significance. Conclusions This meta-analysis supports a positive association of HPgV viremia with lymphoma risk, overall and for the major lymphoma subtypes.

We evaluated the association of Human Pegivirus (HPgV) viraemia with risk of developing lymphoma, overall and by major subtypes. Because this virus has also been associated with better prognosis in the setting of co-infection with human immunodeficiency virus, we further assessed the association of HPgV with prognosis. We used risk factor data and banked plasma samples from 2094 lymphoma cases newly diagnosed between 2002 and 2009 and 1572 frequency-matched controls. Plasma samples were tested for HPgV RNA by reverse transcription polymerase chain reaction (RT-PCR), and those with RNA concentrations

HIV vaccine development is focused on designing immunogens and delivery methods that elicit protective immunity. We evaluated a combination of adenovirus (Ad) vectors expressing HIV 1086.C (clade C) envelope glycoprotein (Env), SIV Gag p55, and human pegivirus GBV-C E2 glycoprotein. We compared replicating simian (SAd7) with nonreplicating human (Ad4) adenovirus-vectored vaccines paired with recombinant proteins in a novel prime-boost regimen in rhesus macaques, with the goal of eliciting protective immunity against SHIV challenge. In both vaccine groups, plasma and buccal Env-specific IgG, tier 1 heterologous neutralizing antibodies, and antibody-dependent cell-mediated viral inhibition were readily generated. High Env-specific T cell responses elicited in all vaccinees were significantly greater than responses targeting Gag. After three intrarectal exposures to heterologous tier 1 clade C SHIV, all 10 sham-vaccinated controls were infected, whereas 4/10 SAd7- and 3/10 Ad4-vaccinated macaques remained uninfected or maintained tightly controlled plasma viremia. Time to infection was significantly delayed in SAd7-vaccinated macaques compared to the controls. Cell-associated and plasma virus levels were significantly lower in each group of vaccinated macaques compared to controls; the lowest plasma viral burden was found in animals vaccinated with the SAd7 vectors, suggesting superior immunity conferred by the replicating simian vectors. Furthermore, higher V1V2-specific binding antibody titers correlated with viral control in the SAd7 vaccine group. Thus, recombinant Ad plus protein vaccines generated humoral and cellular immunity that was effective in either protecting from SHIV acquisition or significantly reducing viremia in animals that became infected, consequently supporting additional development of replicating Ad vectors as HIV vaccines. IMPORTANCE There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV infection and limits in vivo viral replication and associated pathogenesis. Although replicating virus vectors have been advanced as HIV vaccine platforms, there have not been any direct comparisons of the replicating to the nonreplicating format. The present study directly compared the replicating SAd7 to nonreplicating Ad4 vectors in macaques and demonstrated that in the SAd7 vaccine group, the time to infection was significantly delayed compared to the control group, and V1V2 Env-specific binding antibodies correlated with viral outcomes. Viral control was significantly enhanced in vaccinated macaques compared to controls, and in infected SAd7-vaccinated macaques compared to Ad4-vaccinated macaques, suggesting that this vector may have conferred more effective immunity. Because blocking infection is so difficult with current vaccines, development of a vaccine that can limit viremia if infection occurs would be valuable. These data support further development of replicating adenovirus vectors.

To investigate the effects of Cordyceps sinensis (CS) on cellular apoptosis and Sirt1 expression in HK2 cells followed by ischemia-reperfusion (I/R). Methods: HK2 cells were incubated with different concentrations of CS (10, 20, 40, 80, 160, 320 mg/L) for 24 hours, and the optimal concentration of CS was selected by measuring cell proliferation. The confluent HK2 cells were incubated with 0.01 μmol/L antimycin A for 2 hours to induce ischemia in vitro, and then the reperfusion was achieved by incubating cells with glucose-replete complete growth medium for 24 hours. HK2 cells were divided into 4 groups: a control group, an I/R group, an I/R+CS (160 mg/L) group, and an I/R+CS (160 mg/L)+Sirtinol (25 μmol/L) group. Twenty-four hours later, total RNA and protein were collected. The cell proliferation was evaluated by MTT assay; the mRNA and protein expression of Sirt1 and the cleaved caspase-3 were measured by qRT-PCR and Western blot, respectively. The cellular apoptosis rate was determined by Annexin V-FITC/PI double staining and flow cytometry. Results: Certain concentrations (10-160 mg/L) of CS did not show effect on the proliferation of HK2 cells (P0.05), while 320 mg/L of CS inhibited cell proliferation significantly (P0.01); compared with the control group, the mRNA and protein expressions of Sirt1 and the cleaved caspase-3 in the I/R group were up-regulated (P0.01) and the apoptosis rate was extremely high; compared with the I/R group, CS significantly up-regulated Sirt1 mRNA and protein expression (P0.01) while down-regulated cleaved caspase-3 mRNA and protein levels (P0.01), and reduced apoptosis rate (P0.05). The effects of CS were blocked in the presence of sirtinol, an inhibitor of CS. Conclusion: CS protects HK2 cells from I/R injury through activation of Sirt1 pathway.目的：研究冬虫夏草(Cordyceps sinensis，CS)对缺血-再灌注损伤(ischemia reperfusion injury，IRI)的HK2细胞凋亡水平和Sirt1表达的影响，探讨CS对HK2细胞IRI的保护机制。方法：不同浓度CS(10，20，40，80，160，320 mg/L)与HK2细胞共培养24 h，测定细胞增殖率，确定其最佳干预浓度；体外培养的HK2细胞用0.01 μmol/L抗霉素A处理模拟缺血过程，2 h后恢复含血清培养基模拟再灌注过程。将HK2细胞分为对照组，I/R组，I/R+CS组(160 mg/L)，I/R+CS(160 mg/L)+sirtinol(25 μmol/L)组，24 h后提取各组细胞总RNA和蛋白。四甲基偶氮唑盐(MTT)比色法检测细胞增殖；qRT-PCR及Western印迹检测Sirt1和凋亡相关基因(cleaved caspase-3)mRNA及蛋白表达的变化，AnnexinV-FITC/PI双染法及流式细胞仪检测细胞凋亡率。结果：CS 10~160 mg/L与HK2细胞作用24 h，对细胞增殖影响不明显(P0.05)；当浓度增大到320 mg/L时，出现明显抑制HK2细胞增殖的现象(P0.01)。与对照组相比，I/R组Sirt1，cleaved caspase-3 mRNA和蛋白表达增加(P0.01)，细胞凋亡率明显增加(P0.01)；相对于I/R组，I/R+CS组Sirt1 mRNA和蛋白水平增加(P0.01)，而cleaved caspase-3 mRNA和蛋白表达明显下降(P0.01)，细胞凋亡率降低(P0.05)。给予Sirt1抑制剂sirtinol后，I/R+CS+sirtinol(25 μmol/L)组Sirt1 mRNA和蛋白明显降低(P0.05)，cleaved caspase-3 mRNA和蛋白表达明显升高(P0.05)，细胞凋亡率较I/R+CS组增加(P0.05)。结论：CS对HK2细胞IRI具有保护作用，其机制可能与CS促进Sirt1表达有关。.

The Flavivirus genus within the Flaviviridae family is comprised of many important human pathogens including yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZKV), all of which are global public health concerns. Although the related flaviviruses hepatitis C virus and human pegivirus (formerly named GBV-C) interfere with T-cell receptor (TCR) signaling by novel RNA and protein-based mechanisms, the effect of other flaviviruses on TCR signaling is unknown. Here, we studied the effect of YFV, DENV, and ZKV on TCR signaling. Both YFV and ZKV replicated in human T cells in vitro; however, only YFV inhibited TCR signaling. This effect was mediated at least in part by the YFV envelope (env) protein coding RNA. Deletion mutagenesis studies demonstrated that expression of a short, YFV env RNA motif (vsRNA) was required and sufficient to inhibit TCR signaling. Expression of this vsRNA and YFV infection of T cells reduced the expression of a Src-kinase regulatory phosphatase (PTPRE), while ZKV infection did not. YFV infection in mice resulted in impaired TCR signaling and PTPRE expression, with associated reduction in murine response to experimental ovalbumin vaccination. Together, these data suggest that viruses within the flavivirus genus inhibit TCR signaling in a species-dependent manner.

T cell signaling is required for activation of both natural and therapeutic T cells including chimeric antigen receptor (CAR) T cells. Identification of novel factors and pathways regulating T cell signaling may aid in development of effective T cell therapies. In resting human T cells, the majority of Src-family of tyrosine kinases (SFKs) are inactive due to phosphorylation of a conserved carboxy-terminal tyrosine residue. Recently, a pool of enzymatically active SFKs has been identified in resting T cells; however, the significance of these is incompletely understood. Here, we characterized the role of active SFKs in resting human T cells. Pharmacologic inhibition of active SFKs enhanced distal TCR signaling as measured by IL-2 release and CD25 surface expression following TCR-independent activation. Mechanistically, inhibition of the active pool of SFKs induced nuclear translocation of NFAT1, and enhanced NFAT1-dependent signaling in resting T cells. The negative regulation of NFAT1 signaling was in part mediated by the Src-kinase Lck as human T cells lacking Lck had increased levels of nuclear NFAT1 and demonstrated enhanced NFAT1-dependent gene expression. Inhibition of active SFKs in resting primary human T cells also increased nuclear NFAT1 and enhanced NFAT1-dependent signaling. Finally, the calcineurin inhibitor FK506 and Cyclosporin A reversed the effect of SFKs inhibition on NFAT1. Together, these data identified a novel role of SFKs in preventing aberrant NFAT1 activation in resting T cells, and suggest that maintaining this pool of active SFKs in therapeutic T cells may increase the efficacy of T cell therapies.

GBV-C, a pan-lymphotropic flavivirus capable of persistent infection, is associated with prolonged survival and reduced T cell activation in HIV-infected subjects. GBV-C was associated with reduced CD56brt/CD16− NK cell and monocyte activation, and a trend towards reduced B cell activation by measuring cell surface activation markers or HIV entry coreceptors. The GBV-C association was independent of HIV VL. Thus, GBV-C may influence non-T cell immune activation in individuals with HIV infection.

Viruses enter into complex interactions within human hosts, leading to facilitation or suppression of each other’s replication. Upon coinfection, GB virus C (GBV-C) suppresses HIV-1 replication in vivo and in vitro, and GBV-C coinfection is associated with prolonged survival in HIV-infected people. GBV-C is a lymphotropic virus capable of persistent infection. GBV-C infection is associated with reduced T cell activation in HIV-infected humans, and immune activation is a critical component of HIV disease pathogenesis. We demonstrate that serum GBV-C particles inhibited activation of primary human T cells. T cell activation inhibition was mediated by the envelope glycoprotein E2, because expression of E2 inhibited TCR-mediated activation of Lck. The region on the E2 protein was characterized and revealed a highly conserved peptide motif sufficient to inhibit TCR-mediated signaling. The E2 region contained a predicted Lck substrate site, and substitution of an alanine or histidine for the tyrosine reversed TCR-signaling inhibition. GBV-C E2 protein and a synthetic peptide representing the inhibitory amino acid sequence were phosphorylated by Lck in vitro. The synthetic peptide also inhibited TCR-mediated activation of primary human CD4+ and CD8+ T cells. Extracellular microvesicles from GBV-C E2–expressing cells contained E2 protein and inhibited TCR signaling in bystander T cells not expressing E2. Thus, GBV-C reduced global T cell activation via competition between its envelope protein E2 and Lck following TCR engagement. This novel inhibitory mechanism of T cell activation may provide new approaches for HIV and immunoactivation therapy.

GB virus type C (GBV-C) is a lymphotropic virus that can cause persistent infection in humans. GBV-C is not associated with any disease, but is associated with reduced mortality in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Related viruses have been isolated from chimpanzees (GBV-Ccpz) and from New World primates (GB virus type A, GBV-A). These viruses are also capable of establishing persistent infection. We determined the nucleotide sequence encoding the envelope glycoprotein (E2) of two GBV-Ccpz isolates obtained from the sera of captive chimpanzees. The deduced GBV-Ccpz E2 protein differed from human GBV-C by 31 % at the amino acid level. Similar to human GBV-C E2, expression of GBV-Ccpz E2 in a tet-off human CD4+ Jurkat T-cell line significantly inhibited the replication of diverse HIV-1 isolates. This anti-HIV-replication effect of GBV-Ccpz E2 protein was reversed by maintaining cells in doxycycline to reduce E2 expression. Previously, we found a 17 aa region within human GBV-C E2 that was sufficient to inhibit HIV-1. Although GBV-Ccpz E2 differed by 3 aa differences in this region, the chimpanzee GBV-C 17mer E2 peptide inhibited HIV-1 replication. Similarly, the GBV-A peptide that aligns with this GBV-C E2 region inhibited HIV-1 replication despite sharing only 5 aa with the human GBV-C E2 sequence. Thus, despite amino acid differences, the peptide region on both the GBV-Ccpz and the GBV-A E2 protein inhibit HIV-1 replication similar to human GBV-C. Consequently, GBV-Ccpz or GBV-A infection of non-human primates may provide an animal model to study GB virus–HIV interactions.

GB virus C (GBV-C) is a common, non-pathogenic human virus that infects lymphocytes. Persistent GBV-C infection of humans with coexistent human immunodeficiency virus (HIV) infection is associated with prolonged survival, and GBV-C replication inhibits HIV replication in vitro. A GBV-C virus variant was identified in chimpanzees in 1998 and was named GBV-C(trog) or GBV-C(cpz). The prevalence and natural history of GBV-C in chimpanzees remains uncharacterized. We examined the sera from 235 captive chimpanzees for the presence of GBV-C viraemia, viral persistence and clearance, E2 antibody kinetics and RNA sequence diversity. Sequences from six isolates shared more sequence identity with GBV-C(cpz) than with human GBV-C. The prevalence of GBV-C(cpz) viraemia and E2 antibody in chimpanzees (2.5 and 11 %, respectively) was similar to human GBV-C prevalence in healthy human blood donors (1.8 and 9 %, respectively). Persistent GBV-C(cpz) infection occurred in two of the six viraemic animals and was documented for 19 years in one animal. Host subspecies troglodyte GBV-C isolates and published verus GBV-C isolates shared a high degree of sequence identity, suggesting that GBV-C in chimpanzees should be identified with a chimpanzee designation (GBV-C(cpz)). The prevalence and natural history of chimpanzee GBV-C variant (GBV-C(cpz)) appears to be similar to human GBV-C infection. The chimpanzee could serve as an animal model to study HIV-GBV-C co-infection.

Viral infections alter host cell homeostasis and this may lead to immune evasion and/or interfere with the replication of other microbes in coinfected hosts. Two flaviviruses are associated with a reduction in HIV replication or improved survival in HIV-infected people (dengue virus (DV) and GB virus type C (GBV-C)). GBV-C infection and expression of the GBV-C nonstructural protein 5A (NS5A) and the DV NS5 protein in CD4+ T cells inhibit HIV replication in vitro. To determine whether the inhibitory effect on HIV replication is conserved among other flaviviruses and to characterize mechanism(s) of HIV inhibition, the NS5 proteins of GBV-C, DV, hepatitis C virus, West Nile virus, and yellow fever virus (YFV; vaccine strain 17D) were expressed in CD4+ T cells. All NS5 proteins inhibited HIV replication. This correlated with decreased steady-state CD4 mRNA levels and reduced cell surface CD4 protein expression. Infection of CD4+ T cells and macrophages with YFV (17D vaccine strain) also inhibited HIV replication and decreased CD4 gene expression. In contrast, mumps virus was not inhibited by the expression of flavivirus NS5 protein or by YFV infection, and mumps infection did not alter CD4 mRNA or protein levels. In summary, CD4 gene expression is decreased by all human flavivirus NS5 proteins studied. CD4 regulation by flaviviruses may interfere with innate and adaptive immunity and contribute to in vitro HIV replication inhibition. Characterization of the mechanisms by which flaviviruses regulate CD4 expression may lead to novel therapeutic strategies for HIV and immunological diseases.

Objective Interleukin-2 (IL-2) is a cytokine with multiple effects on lymphocytes including induction of CD4 T-cell proliferation. IL-2 administration has been shown to increase CD4 cell counts in HIV-infected people receiving antiretroviral therapy. GB virus C (GBV-C) is an apparently nonpathogenic flavivirus that replicates in CD4 T cells and inhibits HIV replication in vitro by mechanisms including downregulation of HIV entry coreceptors (CCR5 and CXCR4) and induction of chemokines (RANTES, MIP-1alpha, MIP-1 beta, and SDF-1). GBV-C replication is significantly inhibited in vitro by activation of primary CD4 cell cultures with IL-2 and phytohemagglutinin. We sought to determine if there is an interaction between GBV-C and IL-2 in vivo. Methods GBV-C viremia status was characterized in 92 HIV-infected individuals participating in a randomized trial of IL-2 and antiretroviral therapy [AIDS Clinical Trials Group Study (ACTG) 328]. Changes in CD4 cell counts and HIV RNA levels in individuals assigned IL-2 were compared with those in individuals assigned antiretroviral therapy alone. Results Individuals lacking GBV-C viremia had a significantly greater rise in CD4 cell count with IL-2, compared with GBV-C viremic individuals (by 511 cells/microl at week 84; interaction P = 0.02): GBV-C viremic individuals assigned IL-2 did not demonstrate a significant increase in CD4 cell count compared with individuals not assigned to receive IL-2 (95% CI for difference -255 to 397 cells/microl). Conclusion GBV-C viremia was associated with a block in CD4 cell expansion following IL-2 therapy in the ACTG 328 study, and GBV-C status may be an important factor in IL-2 treatment response.

Human Pegivirus (HPgV, formally GB virus C) infects lymphocytes and NK cells in vivo, and infection is associated with reduced T cell and NK cell activation in HIV-infected individuals. The mechanism by which HPgV inhibits NK cell activation has not been assessed. Following IL-12 stimulation, IFNγ expression was lower in HIV-HPgV co-infected subjects compared to HIV mono-infected subjects (p=0.02). In addition, HPgV positive human sera, extracellular vesicles containing E2 protein, recombinant E2 protein and synthetic E2 peptides containing a predicted Tyk2 interacting motif inhibited NK cell IL-12-mediated IFNγ release. E2 protein also inhibited Tyk2 activation following IL-12 stimulation. In contrast, cytolytic NK cell function was not altered by HPgV. Inhibition of NK cell-induced proinflammatory/antiviral cytokines may contribute to both HPgV persistence and reduced immune activation during HIV-coinfection. Understanding mechanisms by which HPgV alters immune activation may contribute towards novel immunomodulatory therapies to treat HIV and inflammatory diseases.

GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.

GB virus type C (GBV-C) is an apparently nonpathogenic virus that replicates in T and B lymphocytes and is a common cause of persistent human infection. Among HIV-1-infected individuals, persistent coinfection with GBV-C is associated with prolonged survival, and infection of blood mononuclear cells or CD4 + T cells with GBV-C and HIV in vitro results in significantly reduced HIV-1 replication. To date, the viral protein(s) that lead to HIV inhibition have not been identified. The GBV-C nonstructural phosphoprotein (NS5A) is predicted to have pleotropic effects on cells, including interactions with the IFN-induced dsRNA-activated protein kinase (PKR). We studied GBV-C NS5A to determine whether it is involved in inhibition of HIV replication. GBV-C NS5A protein from an isolate that was cleared by IFN therapy did not inhibit PKR, whereas NS5A from an isolate that was not cleared by IFN-inhibited PKR function in a yeast genetic system. Both of these GBV-C NS5A proteins were expressed in a CD4 + T cell line (Jurkat), and both induced a potent, dose-dependent inhibition of HIV-1 replication, thus the effect was independent of PKR inhibition. NS5A induced the release of the chemokine SDF-1 and decreased surface expression of the HIV coreceptor CXCR4, potentially explaining the HIV inhibition. Deletion mapping of the NS5A protein found that an 85-aa region between amino acids 152 and 237 inhibits HIV-1 replication. Thus, GBV-C NS5A protein alters the cellular milieu necessary for HIV-1 replication and may provide a previously undescribed therapeutic approach for anti-HIV therapy.

GB virus C (GBV-C) is a common, apparently nonpathogenic human flavivirus that has been associated in some studies with prolonged survival in human immunodeficiency virus (HIV)-positive persons. There are 5 distinct GBV-C genotypes localized to specific geographic regions, and genotype 2 has been previously shown to inhibit HIV replication in vitro in lymphocyte cultures. We identified GBV-C virus isolates representing genotypes 1, 2, and 5 in samples from South African blood donors. GBV-C genotype 1 and 5 isolates replicated in lymphocyte culture, inhibited X4 and R5 HIV-1 isolates, and induced RANTES and stromal-derived factor-1 chemokines in vitro. Thus, African GBV-C genotypes can inhibit HIV replication in vitro.

GB virus type C (GBV-C) causes persistent infection in humans, although the mechanism by which the virus avoids clearance by the host is unknown. To determine if amino acid polymorphisms in the GB virus type C (GBV-C) NS5A and E2 proteins alter response to interferon (IFN) therapy, we studied the sequence of GBVC NS5A and E2 obtained from people receiving IFN therapy. In addition, we expressed recombinant GBVC NS5A protein to determine if it interferes with RNA-activated protein kinase (PKR) function in vitro. GBVC NS5A amplified from a person whose virus was cleared by IFN therapy (IFN sensitive) demonstrated unique amino acid changes occurring in the region that aligns with the hepatitis C virus (HCV) IFN sensitivity-determining region (ISDR) compared with NS5A sequences from individuals who did not clear GBV-C (IFN resistant). There were no differences in the E2 sequences obtained from IFN-sensitive and IFN-resistant isolates. Using a yeast genetic system, IFN-resistant NS5A inhibited PKR-mediated phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) in yeast, whereas IFN-sensitive NS5A did not inhibit PKR function. GBV-C NS5A amino acid polymorphisms appear to be involved in response to IFN therapy, and IFN-resistant GBV-C NS5A inhibited PKR-mediated eIF2alpha phosphorylation in a yeast genetic system, suggesting a mechanism by which GBV-C may evade clearance by naturally occurring host antiviral responses.

Although the median time from human immunodeficiency virus (HIV) infection to the development of malignancies or opportunistic infections defined as AIDS is 7 to 10 years (4), AIDS may develop within a year of infection or it may not develop for more than 20 years following documented HIV seroconversion. Numerous studies have identified host and viral factors that are associated with the HIV disease course, including host HLA types and genetic polymorphisms of genes encoding HIV coreceptor or coreceptor ligands (reviewed in reference 8). Recently, studies found that HIV-positive people who are coinfected with a common, nonpathogenic human flavivirus referred to as GB virus type C (GBV-C) survive significantly longer than do HIV-positive individuals without GBV-C infection (10).

Summary Background People coinfected with HIV and GB virus C (GBV-C) have lower mortality than HIV-positive individuals without GBV-C infection. HIV uses either of the chemokine receptors CCR5 and CXCR4 for entry into CD4-positive cells. Longer survival in HIV-positive individuals is associated with high serum concentrations of ligands for CCR5 (RANTES [regulated on activation, normal T-cell expressed and secreted] and macrophage inflammatory proteins [MIP] 1� and 1� ) and CXCR4 (stromal-derived factor [SDF-1]), and with decreased expression of CCR5 on lymphocytes. Methods Peripheral-blood mononuclear cells were coinfected with GBV-C and HIV, and HIV replication was monitored by measuring infectivity and HIV p24 antigen production. Chemokine secretion was measured by ELISA, chemokinereceptor expression by flow cytometry, and cellular chemokine mRNA expression by differential hybridisation. Findings GBV-C infection of peripheral-blood mononuclear cells resulted in decreased replication of both clinical and laboratory HIV strains that use either CCR5 or CXCR4 as their coreceptor. Inhibition was related to the dose and timing of the GBV-C infection. Expression of mRNA for RANTES, MIP1� , MIP-1� , and SDF-1 and secretion of the chemokines into culture supernatants were higher in GBV-C-infected cells than in mock-infected cells. The inhibitory effect of GBV-C on HIV replication was blocked by incubation with neutralising antibodies against the relevant chemokines, and surface expression of CCR5 was significantly lower in GBV-C-infected cells than in mock-infected cells. Interpretation GBV-C induces HIV-inhibitory chemokines and reduces expression of the HIV coreceptor CCR5 in vitro. This study provides insight into the epidemiological association between GBV-C infection and longer survival in HIV-infected individuals.

GB virus C (GBV-C), which is not known to be pathogenic in humans, replicates in lymphocytes, inhibits the replication of human immunodeficiency virus (HIV) in vitro, and has been associated with a decreased risk of death among HIV-positive persons in some, but not all, studies. Previous studies did not control for differences in the duration of HIV or GBV-C infection.We evaluated 271 men who were participants in the Multicenter Acquired Immunodeficiency Syndrome Cohort Study for GBV-C viremia (by means of a reverse-transcriptase-polymerase-chain-reaction assay) or E2 antibody (by means of an enzyme-linked immunosorbent assay) 12 to 18 months after seroconversion to positivity for HIV (the early visit); a subgroup of 138 patients was also evaluated 5 to 6 years after HIV seroconversion (the late visit).GBV-C infection was detected in 85 percent of men with HIV seroconversion on the basis of the presence of E2 antibody (46 percent) or GBV-C RNA (39 percent). Only one man acquired GBV-C viremia between the early and the late visit, but 9 percent of men had clearance of GBV-C RNA between these visits. GBV-C status 12 to 18 months after HIV seroconversion was not significantly associated with survival; however, men without GBV-C RNA 5 to 6 years after HIV seroconversion were 2.78 times as likely to die as men with persistent GBV-C viremia (95 percent confidence interval, 1.34 to 5.76; P=0.006). The poorest prognosis was associated with the loss of GBV-C RNA (relative hazard for death as compared with men with persistent GBV-C RNA, 5.87; P=0.003).GBV-C viremia was significantly associated with prolonged survival among HIV-positive men 5 to 6 years after HIV seroconversion, but not at 12 to 18 months, and the loss of GBV-C RNA by 5 to 6 years after HIV seroconversion was associated with the poorest prognosis. Understanding the mechanisms of interaction between GBV-C and HIV may provide insight into the progression of HIV disease.

GB virus C/hepatitis G virus (GBV-C) replication in vitro is inefficient and inconsistent. In this study, clinical isolates of GBV-C were evaluated using peripheral blood mononuclear cell (PBMC) based culture methods. Isolates varied consistently in their ability to persistently replicate, and yield increased in cells grown without PHA/IL-2 stimulation. The deduced polyprotein sequence of an isolate that replicated well was determined (GenBank AY196904) and compared to 20 full-length GBV-C sequences. Fourteen of the 16 unique amino acid polymorphisms identified were in the coding regions for nonstructural proteins associated with interferon resistance and RNA replication. These data indicate that clinical GBV-C isolates vary in their ability to persist in culture, do not require PHA/IL-2 stimulation, and that sequence variability in key regulatory regions may affect growth in PBMC cultures. Since GBV-C appears to inhibit HIV replication in a coinfection model, these studies should facilitate determination of the mechanism of this interaction.

GB virus C (GBV-C, also known as hepatitis G virus) commonly causes human infection. Genetically, it is closely related to hepatitis C virus, but GBV-C appears to grow primarily in lymphocytes, not hepatocytes. Although it causes persistent infection in about 25% to 50% of infected individuals, numerous studies have failed to connect GBV-C with any disease process. GBV-C is transmitted sexually, parenterally, and vertically, and due to these shared modes of transmission, coinfection is common among HIV-infected individuals. Of 10 studies done of HIV-GBV-C coinfection, eight found a beneficial effect of GBV-C viremia on HIV-related mortality or response to therapy. The mechanism by which GBV-C may improve survival of HIV-positive people is not known; however, in vitro studies suggest that GBV-C inhibits HIV replication, and preliminary data also point toward alterations in cytokine and/or chemokine expression by GBV-C-infected cells.

Hepatitis C virus (HCV) is difficult to study due to the lack of an efficient cell culture system or small animal model. As a result, HCV–cell interactions are not well-defined. In addition, several studies have identified a subset of patients in whom HCV RNA is present, but HCV antibody is not detected. We produced recombinant baculoviruses that expressed HCV structural proteins (core, E1 and E2, nt 342–2651) or control proteins. The HCV structural protein precursor was processed into immunoreactive proteins of appropriate size, and sucrose density sedimentation and electron microscopy of infected cell lysates demonstrated particle formation. To evaluate HCV antigenicity, particularly in patients who tested negative for HCV antibody in commercial HCV immunoassays but had persistent viremia, we evaluated the virus-like particles (VLPs) in solid-phase immunoassays. VLPs reacted with sera from HCV antibody positive subjects in these solid phase immunoassays, but not with control sera. Plasma samples from 19% (5/26) of HCV antibody negative subjects who were persistently HCV RNA positive also reacted with the HCV VLPs. When incubated with MOLT-4 cells at 4°C, HCV VLPs demonstrated cell binding, and behaved similar to plasma-derived HCV preparations in a flow cytometry-based cell binding assay. These data suggest that recombinant HCV VLPs may allow identification of HCV antibody in patients, including some patients with persistent viremia and who are seronegative with current assays. In addition, HCV VLPs seem useful for evaluating HCV–cell interactions. J. Med. Virol. 68:537–543, 2002. © 2002 Wiley-Liss, Inc.

GB virus type C (GBV-C) is a member of the hepacivirus genus within the Flaviviradae. Persistent GBV-C infection is common in humans, yet it remains unclear if GBV-C causes any disease. Although GBV-C infection has been associated with acute non-A to non-E post-transfusion hepatitis, it does not appear to cause chronic hepatitis. GBV-C is closely related to hepatitis C virus (HCV), but indirect evidence suggests that it does not encode a core protein at the amino terminus of the open reading frame (ORF). This has led to speculation that GBV-C does not have a nucleocapsid. We evaluated the buoyant density of GBV-C, and found very low density particles consistent with virions, and intermediate density particles consistent with nucleocapsids in GBV-C-infected people. In addition, electron microscopy demonstrated an apparent nucleocapsid within an enveloped particle. Although these biophysical data strongly suggest that GBV-C utilizes a nucleocapsid, they do not indicate the origin of the protein content of this particle. To assess this, we evaluated patient plasma for reactivity with a synthetic oligopeptide representing a conserved region near the amino terminus of the predicted ORF. Specific antibody was detected in some individuals, similar to data of Feucht et al. who identified antibody against a recombinant core protein in GBV-C-infected people. These data indicate that GBV-C particles contain nucleocapsids. At least in some patients, the region upstream of the GBV-C E1 protein coding region appears to be expressed, and this region may represent the structural protein of the nucleocapsid.

Human immunodeficiency virus (HIV) disease progression is associated with a helper T cell 1 (Th1) to helper T cell 2 (Th2) cytokine profile switch. Persistent GB virus type C (GBV-C) infection is associated with survival and a serum Th1 cytokine profile in HIV-infected individuals. We found that GBV-C infection increased gene expression of Th1 cytokines and decreased Th2 cytokine expression in peripheral blood mononuclear cells. Furthermore, expression of GBV-C NS5A protein in a CD4(+) cell line resulted in upregulation of Th1 cytokines (tumor necrosis factor α) and downregulation of Th2 cytokines (interleukin 4, interleukin 5, interleukin 10, interleukin 13). GBV-C-induced modulation in T-cell cytokines may contribute to the beneficial effect of GBV-C in HIV-infected individuals.

Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs.